Vaccine development is a critical health care initiative and the development of immunological memory is key to the efficacy of candidate vaccines. To be effective, a vaccine needs to induce, not only a good immune response, but also long-term protective immunity., which is mediated by antigen specific effector and memory T cells. As a population, memory T-cells respond more rapidly to secondary challenge with antigen and are believed to have cellular and molecular characteristics that are distinguishable from effector TH cells. The objective of this proposal is to characterize the development of antigen specific memory TH cells in an experimental mouse model of TH cell immunity. A better understanding of the cellular and molecular basis of memory TH cell development in vivo will provide the means to augment, specifically modify and better direct vaccine-induced immunity. Antigen-specific TH cells have been very difficult to study in normal animals. Primarily, this is due to the extremely low numbers of TH cells that are specific for any one antigen. While it is possible to isolate populations of cells that contain an antigen specific component, these populations are not pure and still contain other cells that confound the unambiguous assay of function. To further complicate analysis, these regulators of the immune response also exert regulatory effects on each other. Therefore, it is imperative to first purify the specific cells of interest and them analyze them at a single cell level. While the antigen specific population may be pure, the individual cells within the population will be heterogeneous with respect to function. In the current study using multi-parameter flow cytometry and gene amplification of T-cell receptors from single cells, it is possible to purify individual antigen specific TH cells and molecularly characterize their specificity directly at the single cell level. They plan to analyze the development of TCR repertoire and relate structure to function to ascertain the biochemical basis for clonal selection in the helper T-cell compartment. They will assess what regulates cytokine production by helper T-cells in vivo. Next, they will probe the molecular basis for cell death and long-term survival in the emergence of effector cell function and memory cell generation. In the last aim, they will use in vitro assays to reveal changes in the molecular physiology of antigen specific helper T cells to understand more clearly the mechanisms that underpin memory cell differentiation. By comparative analysis of antigen specific primary effector cells, resting memory cells and memory effector cells, they will further understand the cellular and molecular dynamics of these regulators of protective immunity.